Breast cancer is the most common type of cancer in women between the ages of 20 and 59 and responsible for ~40,000 deaths annually. If treated early, five-year survival rates for patients in the U.S. are between 83.6 and 98% but drop to 25% once metastatic breast cancer has developed. Thus, there is a great need to develop more effective treatment strategies for metastatic breast cancer. RNA interference (RNAi) can selectively and persistently decrease the expression of any protein at the mRNA level through the cytosolic localization of sequence-specific RNAi molecules including small, interfering RNA (siRNA), dicer-substrate siRNA (DsiRNA), and microRNA (miRNA). Thus, RNAi molecules have tremendous potential to improve the treatment of cancer where the suppression of a single or multiple proteins can produce a therapeutic effect and/or increase the efficacy of current cancer treatments. Our long-term goal is to improve the treatment of metastatic breast cancer with RNAi. The potencies of RNAi molecules after i.v. administration, however, are insufficient for clinical application. Modifying the sense strand of siRNA with 3'-cholesterol (Chol-siRNA) increases the activity of nuclease-resistant siRNA (*siRNA) in the liver and jejunum of mice after i.v. administration but requires a relatively high dose. Our strong preliminary data show that complexation of Chol-*siRNA or Chol-DsiRNA with PLL30-PEG(5K), a block copolymer of 30 poly-L- lysines and 5 kDa polyethylene glycol, greatly increases potency in primary murine syngeneic breast tumors after i.v. administration at a much lower dose. Chol-DsiRNA polyplexes, however, have higher loading, protect Chol-DsiRNA from degradation in serum more effectively, and suppress mRNA in primary murine syngeneic breast tumors for a longer duration than Chol-*siRNA polyplexes. Thus, the overall objectives of this one year proposal are to (i.) provide an initial assessment of the effect of complexation with PLL30-PEG(5K) on the toxicity, PK, and distribution of Chol-DsiRNA after i.v. administration and (ii.) obtain proof-of-concept data that complexation with PLL30-PEG(5K) is likely to increase the potency of Chol-DsiRNA against therapeutically relevant mRNA targets in primary breast tumors and metastases in early and late stage disease. We will accomplish our objectives through the following Specific Aims: (SA1) Determine the effect of complexation with PLL30-PEG(5K) on the toxicity, pharmacokinetics, and distribution of Chol-DsiRNA. The objective of this aim is to provide an initial assessment of the toxicity, PK, and distribution of Chol-DsiRNA after i.v. administration of Chol-DsiRNA polyplexes in a small animal model. (SA2) Determine the activity of Chol-DsiRNA polyplexes in primary murine breast tumors and metastases. The objective of this aim is to obtain proof-of-concept data that Chol-DsiRNA polyplexes are likely to be active against therapeutically relevant mRNA targets in primary breast tumors and metastases. Our approach is innovative because it is a simple and feasible strategy to increase the potency of Chol-DsiRNA in target cells relevant to metastatic breast cancer including microvascular endothelial and tumor epithelial cells.